On the Bactericidal Action of Levallorphan

Abstract

Levallorphan behaves as a bactericidal agent a t a concentration which equals or exceeds the minimal value to block Escherichia coli growth ; the lethal effect of levallorphan results from its action on the plasmic membrane as indicated by permeability changes and lysis of spheroplasts. On whole cells, Mg2+ and Ca2+ loosely bound to the cell surface prevent the penetration of levallorphan and thus act as protecting agents. I n the present communication, we report the bactericidal properties of levallorphan in the absence of growth and consider the possible relationship to the irreversible damage of the protoplasmic membrane. MATERIAL AND METHODSGrowth Conditions E. coli K,,S F-was grown under conditions pre- Survival ExperimentsExponentially-growing bacteria were harvested when their concentration reached approximately 5 x lo8 cells/ml by filtration on Millipore filters (HAWP 0.45 p), and washed a t 37 "C with saline medium in order to remove external adsorbed Mg2+. They were resuspended in the same volume and diluted a thousand times with saline medium. Levallorphan was added, and the mixture was incubated a t 37 "C. Samples were withdrawn, diluted a t room temperature and the number of viable cells was determined as a function of time, or of levallorphan concentration, by plating on complete agar medium. Leakage of Cell ConstituentsThe release of ultraviolet absorbing material was followed in the suspending buffer after centrifugation. The loss of RNA and protein was measured after labelling with [3H]uracil and [14C]leucine. The leakage of Mg2+ in the suspending buffer was determined by atomic absorption spectrophotometry using a Techtron AA4. Obtention of SpheroplastsLysozyme and lysozyme-EDTA spheroplasts were prepared according to the method of Birdsell and Cota-Robles [9] and penicillin spheroplasts according to Lederberg [lo]. The lysis of spheroplasts suspended in 0.5 M sucrose and incubated a t 37 "C, was followed by turbidimetry a t 546 nm. Microscope examination did not reveal any aggregates. "Optical Effect''Changes in the refractive index of bacteria suspended in NaCl 0.1 to 0.75M were recorded as described by Postgate and Hunter [Ill.

Use of polymyxin B, levallorphan, and tetracaine to isolate novel envelope mutants of Escherichia coli

“…The concentrations of the latter agents were really threshold ones; a slight increase in concentration led to killing of all the cells in the culture. This threshold phenomenon was seen previously with levallorphan effects on E. coli cells (4).…”

“…Simon (41) has isolated mutants resistant to levorphanol, but their properties were not explored. The effect oflevallorphan on bacterial cells seems to be in altering permeability, for spheroplasts lyse in the presence of the drug (4), and this lysis is prevented by Ca2+ and Polymyxin DBIT70 L435 T135 P335 Mg2+. The transport of putrescine in E. coli is inhibited by levorphanol (42), again suggesting the cytoplasmic membrane as a target for these drugs.…”

“…Levallorphan (1,3-hydroxy-N-allyl-morphinan), a morphine antagonist, is bacteriocidal for E. coli and lyses E. coli spheroplasts (4). The agonist levorphanol, which is closely related to levallorphan, leads to a change in the phospholipid composition of E. coli, with an increased turnover of phosphatidyl glycerol and accumulation of cardiolipin (48).…”

The Energy‐Dependent Degradation of Guanosine 5′‐Diphosphate 3′‐Diphosphate in Escherichia coli

“…First, morphinans such as levallorphan, which had no chelating activity on Mn2+ ions but perturbated the organization of the cytoplasmic membrane [53], had a drastic effect on ppGpp breakdown, mimicking a carbon-source-induced downshift [32]. Second, carbon source exhaustions, or energy poisons such as 2,4-dinitrophenol were shown to have the same effect as levallorphan or as the spoTmutation [31].…”

Bacteriological Studies with Morphine-Like Narcotics: Relevance to Narcotic Actions in Mammals?

“…This is accompanied by reduced ribonucleic acid (24), protein (10,24) and deoxyribonucleic acid (10) biosyntheses, alterations in lipid metabolism and composition (27), changes in the transport of certain amino acids (12), polyamines (22) and Mg2+ (7) across membranes, mobilization of (probably membrane associated) adenosine triphosphatase (10), and other interferences with bacterial envelope functions such as the adsorption of bacteriophage (19) or the formation of mating pairs (14). A bactericidal effect of levallorphan at 3.8 x 10-3 M has also been attributed to an action on the bacterial membrane (3). The nonoccurrence of ribonucleic acid biosynthesis in Escherichia coli, exposed to levallorphan, has been studied especially with respect to attending features of biochemical regulation (4,5,11).…”